Nuatigenin-Type Steroidal Saponins from Veronica fuhsii and V. multifida

Nuatigenin-Type Steroidal Saponins from Veronica fuhsii and V. multifida Meltem Ozipek, a Iclal Saracoglu, a Yukio Ogihara b and İhsan Çaliş a,* a Department of Pharmacognosy, Faculty of Pharmacy, Hacettepe University, TR-06100 Ankara, Turkey. Fax: +90-3 12-3 11 47 77. E-mail: icalis@hacettepe.edu.tr b Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Nagoya City University, Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan * Author for correspondence and reprint requests Z. Naturforsch. 57 c, 603Ð608 (2002); received April 8/May 10, 2002 Veronica, Steroidal Glycosides, Multifidoside A new nuatigenin-type steroidal saponin, multifidoside (2), was isolated from the aerial parts of Veronica fuhsii and V. multifida and its structure was identified as 3-O-{[α-lrhamnopyranosyl-(152 glu )]-[β-d-glucopyranosyl-(154 rha )-α-l-rhamnopyranosyl (154 glu )]- β-d-glucopyranosyl}nuatigenin 26-O-β-d-glucopyranoside. Additionally, a known steroidal saponoside, aculeatiside A (1), from V. fuhsii, a phenylethanoid glycoside, verpectoside A (3), and a flavon glycoside, isoscutellarein 7-O-(2 -O-6 -O-acetyl-β-d-allopyranosyl-β-d-glucopyranoside) (4) from V. multifida were isolated. Introduction Veronica species (Scrophulariaceae) contain mainly iridoid glycosides and some phenylethanoid glycosides and flavonoid compounds (Lahloub, 1989; Lahloub et al., 1993; Taskova et al., 1998; Taskova et al., 1999; Chari et al., 1981; Aoshima et al., 1994). However saponins of Veronica species were rarely reported (Tamas, et al., 1984; Bogacheva et al., 1980; Gvazava and Pkhidze, 1988). Our previous studies have shown the presence of iridoid glycosides in Veronica multifida L. (Ozipek et al., 2000) and V. fuhsii FREYN et SINT (Ozipek et al., 1998) as well as phenylethanoid glycosides, plantamajoside and fuhsioside, and a flavone glucoside, luteolin 7-Oglucoside, in the latter plant (Ozipek et al., 1999). We now report a new furospirostanol glycoside, multifidoside (2), from the aerial parts of V. fuhsii and V. multifida besides a known furospirostanol glycoside, aculeatiside A, from V. fuhsii (1). In addition, a phenylethanoid glycoside (3) and a flavone glycoside (4), isolated from V. multifida, are also reported. Compound (3) was identified as verpectoside A (3,4-dihidroxy-β-phenylethoxy-O-[α-larabinopyranosyl-(15 2)]-[α-l-rhamnopyranosyl- (15 3)]-4-O-feruloyl-β-d-glucopyranoside (Saracoglu et al., 2002) and (4) as isoscutellarein 7-O- (2 -O-6 -O-acetyl-β-d-allopyranosyl-β-dglucopyranoside) (Lenherr et al., 1984) from their UV, IR and NMR spectroscopic data by comparison with reported data. Material and Methods General experimental procedures Optical rotation was measured on a Autopol IV Rudolph Research Analytical polarimeter using a sodium lamp operating at 589 nm. IR spectra were measured on a Perkin-Elmer 2000 FT-IR spectrometer in KBr pellets. NMR spectra were recorded on a Joel JNM-A500, Bruker AMX 300 and DRX 500 spectrometers. HR-FAB-MS spectra were obtained on an Ion Spec Ultima FTMS spectrometer. Plant material Veronica fuhsii (Scrophulariaceae) was collected from Kizilcahamam-Isikdagi in May 1988. The voucher specimen (HUEF-88148) has been deposited in the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey. Veronica multifida was collected from Sivas (Yildizeli-Akdag) in June 1998. The voucher specimen (HUEF-98048) has been deposited in the Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey. Extraction and isolation Air-dried aerial parts (220 g) of V. fuhsii were extracted with MeOH (2 1 l). The methanolic extract was evaporated in vacuo. The residue (48 g) 0939Ð5075/2002/0700Ð0603 $ 06.00 2002 Verlag der Zeitschrift für Naturforschung, Tübingen www.znaturforsch.com D

604 M. Ozipek et al. Saponins of Veronica species was dissolved in water and then extracted with petroleum ether and the petroleum ether phase discarded. The aqueous phase was concentrated and chromatographed over polyamide column eluting with H 2 O, followed by increasing concentrations of MeOH to yield four main fractions: A-D [A 1 - A 5, 9.2 g (H 2 O); B, 675 mg (50% MeOH); C, 400 mg (75% MeOH); D, 1 g (MeOH)]. Fraction A 4 (1.4 g) was applied to MPLC using reversed phase material (LiChroprep C 18 ) with increasing amount of MeOH in H 2 O. Fraction eluted with 75% MeOH was rechromatographed over Si gel by stepwise elution with a CHCl 3 -MeOH-H 2 O (80:20:25 70:30:3) solvent system to give 1 (11 mg) and 2 (9 mg). Air-dried aerial parts of V. multifida (100 g) were extracted twice with methanol (each 1 l). The methanolic extract was evaporated in vacuo. The residue (20 g) was dissolved in water and then extracted with petroleum ether and the petroleum ether phase discarded. The remaining aqueous phase was concentrated (12 g) and chromatographed over a polyamide column eluting with H 2 O, followed by increasing concentrations of MeOH to yield four main fractions: AÐD [A, 4.2 g (H 2 O); B 1 ÐB 3, 1.4 g (50% MeOH); C 1 ÐC 3, 1.9 g (75% MeOH); D, 1.3 g (MeOH)]. B 2 (250 mg) was chromatographed over Si gel by stepwise elution with a CH 2 Cl 2 -MeOH (9:15 6:4) solvent system and then rechromatographed over Sephadex LH- 20 with MeOH to give 2 (4 mg) and 3 (1.5 mg). C 2 (225 mg) was chromatographed over Si gel using CH 2 Cl 2 -MeOH-H 2 O (80:20:2 5 60:40:4) solvent system and then rechromatographed over Sephadex LH-20 with MeOH to give 4 (20 mg). Aculeatiside A (1): IR υ max (KBr) cm Ð1 : 3420 (OH), 2929 (CH), 1456, 1039, 915, 860, 820. 1 H NMR (500 MHz, pyridine-d 5 ) (sugar moiety see Table I); δ 4.23 (H-3, overlapped), 5.32 (1H, br s, H-6), 4.72 (1H, t, J = 7.3 Hz, H-16), 1.74 (H-17, overlapped), 4.20 (1H, d, J = 10.0, H-26a), 3.91 (1H, d, J = 10.0, H-26b), 0.81 (3H, s, H-18), 1.05 (3H, s, H-19), 1.08 (3H, d, J = 7.0, H-21), 1.41 (3H, s, H-27). 13 C NMR (125.6 MHz, pyridine-d 5 ), see Table II; HR-FAB-MS: (m/z) 1069.5 [M + Na] + Mulifidoside (2): [α] D 20-78.0 (c 0.1, MeOH) IR υ max (KBr) cm Ð1 : 3418 (OH), 2927(CH), 1456, 1045, 910, 870, 820. 1 H NMR (300, 500 MHz, CD 3 OD) (sugar moiety, see Table I); δ 3.60 (1H, t, J = 9.2, H-3), 5.37 (1H, d, J = 4.9, H-6), 4.45 (1H, t, J = 7.3 Hz, H-16), 1.76 (1H, t, J = 7.0, H-17), 3.86 (1H, d, J = 10.0, H-26a), 3.48 (1H, d, J = 10.0, H-26b), 0.81 (3H, s, H-18), 1.05 (3H, s, H-19), 0.99 (3H, d, J = 7.0, H-21), 1.23 (3H, s, H-27). 13 C NMR (125.6 MHz, CD 3 OD), see Table II; HR-FAB-MS: (m/z) 1231.5 [M + Na] +. Results and Discussion Compound 1 was obtained as a white amorphous powder. The high resolution (HR)-FAB-MS of compound 1 exhibited a pseudomolecular ion peak at m/z 1069.5 [M + Na] + which is compatible with the molecular formula C 51 H 82 O 22, requiring eleven degrees of unsaturation. The 1 H-NMR spectrum revealed the signals for six methyl groups at δ H 0.81 (s, CH 3-18), 1.05 (s, CH 3-19), 1.08 (d, J = 7.0 Hz, CH 3-21), 1.41 (s, CH 3-27), 1.78 (d, J = 6.1 Hz, CH 3-6 ), 1.64 (d, J = 6.1 Hz, CH 3-6 ), an olefinic proton at δ H 5.32 (brs, H-6), and four anomeric protons at δ H 4.95 (d, J = 7.9 Hz), 4.98*, 5.87 (brs) and 6.41 (brs), and a pair of hydroxymethylene protons as an AB system (δ H 4.20 and 3.91, J AB = 10.0 Hz, H 2-26), predicting a tetraglycosidic steroidal structure. Thus, the signals at δ H 1.64 and 1.78 arising from two secondary methyl protons were attributed to two rhamnose units. In the 13 C-NMR spectrum, four anomeric carbon signals were observed at δ C 100.3, 105.5, 102.9 and 102.0, confirming the tetraglycosidic structure. All NMR assignments (Tables I, II) were based on COSY, HMQC and HMBC experiments. The chemical shifts and coupling constants of the signals assigned to the sugar moiety indicated the presence of two glucose and two rhamnose units. The signals observed at δ C 140.7 and 121.9 were assigned to the olefinic carbon atoms. Since this last functional group accounted for one double bond equivalent, 1 was considered as decacyclic. Hence, the aglycon was found to be hexacyclic. The 13 C NMR data contained 27 carbon atoms for the steroidal aglycon. The quaternary carbon signals displayed at δ C 120.3 and 83.9 indicated the dioxygenated C-22 carbon and C-25 of the agylcone, respectively, which are characteristic for the furospirostanol sapogenins (Agrawal et al., 1985). Thus, the signals at δ C 24.4 and 77.5 were assigned to the methyl (C-27) and hydroxymethyl (C-26) groups, respectively. This assumption was also confirmed by the long-range correlations between H 2-26/C-25

M. Ozipek et al. Saponins of Veronica species 605 Fig. 1. Chemical structures of the isolated compounds 2. and H-27/C-25. The remaining carbon and proton signals attributed to the aglycone were in good accordance to those of nuatigenin reported (Saijo et al., 1983; Mimaki and Sashida, 1990). The downfield shifts at δ C 78.1 (C-3) and 77.5 (C-26) showed that the glycosyl residues linked to the hydroxyls at C-3 and C-26 which indicated the bisdesmosidic structure of the compound 1. The HMBC correlations observed between H-1 (δ H 5.87) and C-2 (δ C 78.6); H-1 (δ H 6.41) and C-4 (δ C 77.8); revealed the sites of the linkages of sugar moiety attached to C-3 of the sapogenol. Finally, the remaining glucose unit was found to be linked to the hydroxymethyene group of the sapogenin moiety, which was confirmed by the long-range correlations between H-1 /C-26 visa verse H 2-26/ C-1 in the HMBC experiment. The signals attributed to compound 1 were in good agreement with those of aculeatiside A, a nuatigenin tetraglycoside (Saijo et al., 1983). Therefore the structure of 1 was identified as aculeatiside A, 3-O-[α-lrhamnopyranosyl-(15 2 glu )-α-l-rhamnopyranosyl- (15 4 glu )-β-d-glucopyranosyl] nuatigenin 26-O-βd-glucopyranoside.

606 M. Ozipek et al. Saponins of Veronica species 1* 2 # H δ H ppm, J [Hz] H δ H ppm, J [Hz] Glc-1 4.95 δ (7.9) Glc-1 4.49 δ (7.9) 2 4.23* 2 3.38* 3 4.23* 3 3.57* 4 4.40* 4 3.54 t (7.5) 5 3.65 m 5 3.32* 6 4.23* 6 3.79 dd (12.0/2.0) 4.11* 3.65 dd (12.0/6.0) Rha-1 5.87 brs Rha-1 5.19 δ (1.5) 2 4.84 dd (1.7/3.4) 2 3.92 dd (3.4/1.7) 3 4.64 dd (3.4/9.4) 3 3.67* 4 4.37* 4 3.38* 5 4.98* 5 4.12 dd (10.0/6.1) 6 1.78 δ (6.1) 6 1.33 δ (6.1) Rha-1 6.41 brs Rha-1 4.85 brs 2 4.70 dd (1.7/3.4) 2 3.85 dd (3.4/1.7) 3 4.56* 3 3.67* 4 4.34* 4 3.65* 5 4.95* 5 4.02 dq (10.0/6.1) 6 1.64 δ (6.1) 6 1.24 δ (6.1) Glc-1 4.58 δ (7.9) 2 3.19 dd (7.9/9.0) 3 3.37* 4 3.28* 5 3.26* 6 3.87 dd (12.0/2.0) 3.70 dd (12.0/6.0) Glc-1 4.98* Glc-1 4.30 δ (7.9) 2 4.05 dd (7.9/9.0) 2 3.22 dd (7.9/9.0) 3 4.28* 3 3.37* 4 4.28* 4 3.28* 5 3.96 m 5 3.26* 6 4.56 dd (12.0/2.0) 6 3.87 dd (12.0/2.0) 4.43 dd (16.0/6.0) 3.70 dd (12.0/6.0) Table I. 1 H NMR spectroscopic data for sugar moieties of aculeatiside A (1*) and multifidoside (2 # ) (Pyridine-d 5 *, CD 3 OD # ). * Signal pattern unclear due to overlapping. Compound 2 was also obtained as a white amorphous powder. The HR-FAB-MS of compound 2 exhibited a pseudomolecular ion peak at m/z 1231.5 [M + Na] + which is compatible with the molecular formula C 57 H 92 O 27. The signals observed in 1 H and 13 C NMR spectra were very similar to those of compound 1. The only difference is the signals arising from an additional sugar unit. The 1 H NMR spectrum indicated the signals for six methyl groups at δ H 0.81 (s, CH 3-18), 1.05 (s, CH 3-19), 0.99 (d, J = 7.0 Hz, CH 3-21), 1.23 (s, CH 3-27), 1.33 (d, J = 6.1 Hz, CH 3-6 ), 1.24 (d, J = 6.1 Hz, CH 3-6 ), an olefinic proton at δ H 5.37 (d, J = 4.9), five anomeric protons at δ H 4.49 (d, J = 7.9 Hz), 4.58 (d, J = 7.9 Hz), 4.30 (d, J = 7.9 Hz), 5.19 (d, J = 1.5 Hz), 4.85 (brs), and a pair of hydroxymethylene protons as an AB system (δ H 3.86 and 3.48, J AB = 10.0 Hz, H 2-26), confirming the pentaglycosidic structure of compound 2. The signals at δ H 1.24 and 1.33 arising from two secondary methyl protons were attributed to two rhamnose units. Five anomeric carbon signals observed at δ C 100.4, δ C 105.5, δ C 104.9, δ C 102.2 and δ C 102.6 in the 13 C NMR spectrum verified the pentaglycosidic structure. The complete assignments (Tables I, II) of all proton and carbon resonances were based on the DQF-COSY, HSQS, HMQC and HMBC experiments. The assignments attributed to the sugar moiety indicated two rhamnose and three glucose units. The 13 C NMR spectrum showed 27 carbon atoms for the steroidal aglycone moiety. The chemical shifts and coupling constants of the signals assigned to the aglycone moiety indicated the presence of the same steroidal aglycone,

M. Ozipek et al. Saponins of Veronica species 607 1* 2 # 1* 2 # C DEPT δ C δ C C DEPT δ C C δ C 1 CH 2 37.5 38.5 Glc-1 CH 100.3 Glc-1 100.4 2 CH 2 30.2 30.7 2 CH 78.6 2 79.3 3 CH 78.1 79.2 3 CH 78.0 3 78.0 4 CH 2 39.0 39.4 4 CH 77.8 4 79.6 5 C 140.7 141.8 5 CH 77.0 5 76.6 6 CH 121.9 122.5 6 CH 2 61.3 6 61.9 7 CH 2 32.2 32.8 Rha-1 CH 102.9 Rha-1 102.2 8 CH 31.6 32.8 2 CH 72.5 2 72.1 9 CH 50.3 51.7 3 CH 72.8 3 72.3 10 C 37.1 38.0 4 CH 74.1 4 73.9 11 CH 2 21.1 21.9 5 CH 69.5 5 69.7 12 CH 2 39.8 40.9 6 CH 3 18.7 6 17.9 13 C 40.1 41.5 Rha-1 CH 102.0 Rha-1 102.6 14 CH 56.5 57.7 2 CH 72.6 2 72.2 15 CH 2 32.3 33.1 3 CH 72.7 3 72.3 16 CH 81.0 82.1 4 CH 73.9 4 83.1 17 CH 62.5 63.2 5 CH 70.4 5 69.2 18 CH 3 16.2 16.5 6 CH 3 18.5 6 17.9 19 CH 3 19.4 19.8 CH Glc-1 105.5 20 CH 38.6 39.3 CH 2 76.1 21 CH 3 15.2 15.0 CH 3 78.2 22 C 120.3 121.7 CH 4 71.6 23 CH 2 33.1 33.5 CH 5 78.0 24 CH 2 34.0 33.7 CH 2 6 62.7 25 C 83.9 85.2 Glc-1 CH 105.5 Glc-1 104.9 26 CH 2 77.5 77.5 2 CH 75.4 2 75.2 27 CH 3 24.4 24.2 3 CH 78.5 3 77.7 4 CH 71.6 4 71.4 5 CH 78.6 5 78.1 6 CH 2 62.7 6 62.7 Table II. 13 C NMR spectroscopic data for aculeatiside A (1*) and multifidoside (2 # ) (Pyridine-d 5 *, CD 3 OD # ). nuatigenin, as compound 1. In the HBMC spectrum, the anomeric proton of the glucose (H-1 ) (δ H 4.49) exhibited a longe-range coupling between C-3 of the aglycone (δ C 79.2) whereas H-1 of glucose (δ H 4.30) showed a correlation with C-26 of the agylcone (δ C 77.5) indicating the bisdesmosidic structure of 2. Additionally longe-range correlations observed between H-1 of rhamnose (δ H 5.19) and C-2 of glucose (δ C 79.3); H-1 of rhamnose (δ H 4.85) and C-4 of glucose (δ C 79.6); Fig. 2. Selected HMBC correlations for multifidoside (2).

608 M. Ozipek et al. Saponins of Veronica species H-1 of glucose (δ H 4.58) and C-4 of rhamnose (δ C 83.1), revealing the sites of the interglycosidic linkages in the sugar moiety attached to C-3 of the sapogenol. Consequently, the structure of compound 2 was established as 3-O-{[α-l-rhamnopyranosyl- (15 2 glu )]-[β-d-glucopyranosyl-(15 4 rha )-α-lrhamnopyranosyl (15 4 glu )]-β-d-glucopyranosyl} nuatigenin 26-O-β-d-glucopyranoside. For this novel structure, the trival name multifidoside is proposed by us. Agrawal P. K., Jain D. C., Gupta R. K. and Thakur R. S. Ozipek M., Saracoglu I., Çalis I., Kojima K., and Ogi- (1985), Carbon-13 NMR spectroscopy of steroidal sa- hara, Y. (2000), Catalpol derivative Iridoids from the pogenins and steroidal saponins. Phytochemistry 24, roots of Veronica multifida. Hacettepe University, 2479Ð2496. Journal of Faculty of Pharmacy 20, 1Ð6. Aoshima H., Miyase T. and Ueno A. (1994), Phenyletha- Ozipek M., Saracoglu I., Kojima K., Ogihara Y. and noid glycosides from Veronica persica. Phytochemistry Çalis, I., (1999), Fuhsioside, a new phenylethanoid 37, 547Ð550. glucoside from Veronica fuhsii. Chem. Pharm. Bull. Bogacheva N. G., Stikhin V. A. and Mladentseva, M. S. 47, 561Ð562. (1980), Diosgenin from Veronica teucrium. Khim. Prir. Ozipek M., Saracoglu I., Maruyama M., Takeda T. and Soedin. 3, 423Ð424 (Russ.) Ref.; (1980), CA 93, Çalis, I. (1998), Iridoid glucosides from Veronica fuhsii. :110627 n. Hacettepe University, Journal of Faculty of Pharmacy Chari V. M., Grayer-Barkmeijer R. J., Harborne J. B. and 18,9Ð14. Osterdahl, B. G. (1993), An acylated allose-containing Saijo R., Fuke C., Murakami K., Nohara T. and Tomi- 8-hydroxyflavone glycoside from Veronica filiformis. matsu, T. (1983), Two steroidal glycosides, aculeatiside Phytochemistry 20, 1977Ð1979. A and B from Solanum aculeatissimum. Phytochemis- Gvazava L. N. and Pkheidze T. A. (1988), Steroidal sa- try 22, 733Ð736. ponins of Veronica gentianoides. Khim. Prir. Soedin. Saracoglu I., Harput U. S., Inoue M. and Ogihara, Y. 5, 761Ð762 (Russ.). Ref.; (1989) CA 110, 111766 p. (2002), New phenylethanoid glycosides from Veronica Lahloub M. F. (1989), Iridoid glycosides from Veronica pectinata var. glandulosa and their free radical scavfiliformis. Planta Med. 55, 623. enging activities. Chem. Pharm. Bull. 50, 665Ð668. Lahloub M. F., Zaghloul M. G., Affifi M. S. and Sticher, Tamas M., Rosca M. and Scarlat, M. A. (1984), Studiul O. (1993), Iridoid glucosides from Veronica anagallis- fitochimic al plantei Veronica officinalis L. Clujul aquatica. Phytochemistry 33, 401Ð405. Med. 57, 169Ð172 (Rom.). Lenherr A., Lahloub M. F. and Sticher, O. (1984), Three Taskova R., Handjieva N., Evstatieva L. and Popov, S. flavonoid glycosides containing acetylated allose from (1999), Iridoid glycosides from Plantago cornuti, Plan- Stachys recta. Phytochemistry 23, 2343Ð2345. tago major and Veronica cymbalaria. Phytochemistry Mimaki Y. and Sashida Y. (1990), Steroidal saponins 52, 1443Ð1445. from the bulbs of Lilium brownii. Phytochemistry 29, Taskova R., Handjieva N., Peev D. and Popov, S. (1998), 2267Ð2271. Iridoid glucosides from three Veronica species. Phytochemistry 49, 1323Ð1327.